A re-appraisal of the BHK 21 clone-13 cell transformation system with respect to the assay conditions and the mutational origin of the transformed phenotype.

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Two BHK 21/13 cell lines (BHK-ICI and BHK-A3) were used for studies on cell transformation. The BHK-ICI cells formed micro-colonies in agar while the BHK-A3 cells did not. The size distribution of colonies in agar derived from BHK-ICI cells was homogeneous and did not permit a distinction between untransformed micro-colonies and transformed macro-colonies. Moreover, the transformation frequency in BHK-ICI cells was influenced substantially by cell density, as densities greater than 500 cells/ml reduced the observed transformation frequency, whereas no reduction in transformation frequency was observed for BHK-A3 cells for densities up to 10(4) cells/ml. Therefore, we recommend the use of a BHK cell line that does not form micro-colonies for the transformation assay. In contrast with published data, obtained at high cell density with BHK cells that form micro-colonies, induction of cell transformation in BHK-A3 cells by treatment with 4NQO did not behave as a mutational trait. (i) Continuous treatment did not lead to a steady increase in transformation frequency but to a reduction in transformation frequency after the second day of exposure. (ii) A continuous treatment up to 24 h proved to be about five times more effective in inducing cell transformation than a one hour acute treatment. The results support the suitability of the BHK cells as an assay for cell transformation if the adverse effects of cell density are met and the formation of micro-colonies is prevented. The use of BHK-A3 cells at a density of 10(4)/ml fulfills these requirements.